Structure, function, and disease biology of MICU1/MICU2

摘要 线粒体通过一种称为单一转运蛋白的钙激活通道摄取钙。钙的摄取可以使 细胞器的新陈代谢状态与快速变化的能量需求相匹配，进而调整 神经传递和肌肉收缩等关键过程。单一转运体是钙激活的钙。 受钙结合异二聚体MICU1/MICU2调节的通道。最近，MICU1基因的突变 被确认为一种新形式的肌病的原因，其特征是疲劳和运动不耐受 线粒体肌病的典型特征。MICU1和MICU2感知的精确机制 钙调节单转运体，以及这种异源二聚体中的损伤如何导致这种非常不寻常的肌病 不知道。通过这个对偶PI授予，我们建议：(1)表征化合物的物理化学性质 MICU1和MICU2。在大肠杆菌中表达的野生型蛋白和突变体将受到尊重 对齐聚状态、结构稳定性、钙镁离子结合亲和力和pH敏感性的影响。(2)确定 MICU1和MICU2对单一转运体进行调控的结构基础。高分辨X射线结构的研究 将确定单独的MICU2以及apo和钙结合形式的MICU1/2异二聚体。 电子冷冻显微镜将用于确定MICU1/2的天然类低聚物的结构 很复杂。(3)细胞内线粒体钙动力学研究。使用基因组编辑技术 我们已经设计了一个强大的体内基因敲除细胞系系统，用于研究工程和 人类自然发生的MICU1和MICU2突变对线粒体钙转运动力学和 能量学，以及(4)了解人类MICU1肌病的代谢和生物能量基础 将Micu1/-老鼠作为模型进行研究。我们将描述肌肉组织学特征，线粒体 生物能量学，运动表现和代谢组学，以及单纤维收缩能力，以检验这一假设 MICU1的缺失会导致能量代谢紊乱，从而导致肌病。我们的目标-跨越 从分子到动物生理学--将产生对调节的整体和机制的理解 MICU1和MICU2对线粒体钙摄取的影响及其对一个新发现的人类的贡献 肌病。

ABSTRACT Mitochondria uptake calcium via a calcium activated channel called the uniporter. Calcium uptake allows the organelle's metabolic state to be matched to rapidly changing energy requirements, and, in turn, tune key processes such as neurotransmission and muscle contraction. The uniporter is calcium-activated calcium channel regulated by the calcium binding heterodimer MICU1/MICU2. Mutations in MICU1 have recently been identified as a cause of a new form of a myopathy characterized by fatigue and exercise intolerance without the classical features of mitochondrial myopathy. The precise mechanisms by which MICU1 and MICU2 sense calcium to regulate the uniporter, and how lesions in this heterodimer lead to this highly unusual myopathy are not known. Through this dual PI grant we propose to: (1) Characterize the physicochemical properties of MICU1 and MICU2. The wild type proteins and mutants expressed in E. coli will be characterized with respect to the oligomeric state, structural stability, Ca2+ and Mg2+ binding affinities, and pH sensitivity. (2) Determine the structural basis for the regulation of the uniporter by MICU1 and MICU2. High resolution X-ray structures of MICU2 alone and of the MICU1/2 heterodimer in the apo and Ca2+-bound forms will be determined. Electron cryo-microscopy will be used to determine the structure of a native-like oligomer of MICU1/2 complex. (3) Investigate mitochondrial calcium dynamics in cellular systems. Using genome-editing technology we have engineered a powerful in vivo system of knockout cell lines for studying the effects of engineered and naturally occurring human mutations in MICU1 and MICU2 on the mitochondrial calcium transport kinetics and energetics, and (4) Understand the metabolic and bioenergetic basis of human MICU1 myopathy by investigating the Micu1-/- mouse as a model. We will characterize the muscle histology, mitochondrial bioenergetics, exercise performance and metabolomics, and single fiber contractility to test the hypothesis that loss of MICU1 leads to a myopathy by causing disturbances in energy metabolism. Our aims – spanning the molecular to animal physiology -- will yield a holistic and mechanistic understanding of the regulation of mitochondrial calcium uptake by MICU1 and MICU2 and its contribution to a newly described human myopathy.